Salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase gene as well as expression protein and application of salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase gene

Abstract

The invention discloses a salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase gene, an expression protein and application, the DNA (Deoxyribose Nucleic Acid) sequence of the salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase gene is respectively as shown in SEQ ID NO.1, and the amino acid sequence of the salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase expressed by the salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase gene is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the salt-tolerant ethanol-tolerant beta-1, 3-1, 4-glucanase gene. The beta-1, 3-1, 4-glucanase TaGlu16B disclosed by the invention shows the maximum enzyme activity when the pH value is 5.0 and the temperature is 50 DEG C, and has remarkable pH stability (the enzyme activity is kept about 94%, 72% and 86% respectively when the pH values are 4, 5 and 6). Besides, when the concentration of NaCl in the beta-1, 3-1, 4-glucanase TaGlu16B is 2% and the concentration of NaCl in the beta-1, 3-1, 4-glucanase TaGlu16B is 15%, the activity of the TaGlu16B reaches 116.9% and 113% respectively. In ethanol with the concentration of 0-20%, TaGlu16B can keep the activity of 70% or above, beta-glucan can be effectively degraded, the filtering time in the beta-glucan saccharification process in the brewing industry is shortened, the viscosity in the beta-glucan saccharification process in the brewing industry is reduced, and therefore the food manufacturing cost is effectively reduced, and TaGlu16B is expected to become a catalyst in the food and biological industry.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase gene, an expressed protein and their applications. Background Art

[0002] β-glucan is a glucose polymer linked by β-glycosidic bonds, which widely exists in the cell walls of cereals (such as barley, oats, wheat) and some microorganisms (such as yeast, fungi). The high viscosity property of β-glucan often has an adverse impact on the feed and food processing industries. For example, increasing the wort viscosity in beer brewing will affect its filtration efficiency. Therefore, it is necessary to hydrolyze β-glucan to obtain high-value-added products. The enzymatic hydrolysis method is the main method for hydrolyzing β-glucan, which has the advantages of mild reaction conditions, little environmental pollution and high efficiency.

[0003] β-glucanases that catalyze the hydrolysis of β-glucan include four types: β-1,3-1,4-glucanase (EC3.2.1.73), β-1,4-glucanase (EC 3.2.1.4), β-1,3-glucanase (EC 3.2.1.39) and β-1,3(4)-glucanase (EC 3.2.1.6). Among them, β-1,3-1,4-glucanase decomposes long-chain β-glucan into oligosaccharides and monosaccharides by specifically cleaving the β-1,4-glycosidic bond adjacent to the β-1,3-linkage. At present, β-1,3-1,4-glucanase has been industrially applied, but its performance still has limitations. For example, the degradation efficiency of high-concentration substrates is limited, and it is necessary to increase the enzyme amount, increasing the cost; for example, it cannot exist under acidic, high-concentration salt and ethanol conditions. Especially in the brewing industry, β-glucan is widely used as a raw material in the brewing industry, but its high viscosity and low filtration efficiency will have a negative impact on the brewing quality. Therefore, it is highly desirable to develop a new type of β-1,3-1,4-glucanase with good biochemical properties.

[0004] Therefore, according to the existing problems, it is necessary to develop a highly active β-1,3-1,4-glucanase under high-salt and relatively high ethanol concentration conditions, expand the industrial application range of β-1,3-1,4-glucanase, enable it to more effectively degrade β-glucan, and reduce the cost of food processing. Summary of the Invention

[0005] The purpose of the present invention is to provide a salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase gene, an expressed protein and their applications in view of the deficiencies of the prior art.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] The present invention provides a salt- and ethanol-tolerant β-1,3-1,4-glucanase gene, and the DNA sequence of the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene is respectively as shown in SEQ ID NO.1.

[0008] The present invention also provides a salt- and ethanol-tolerant β-1,3-1,4-glucanase, and its amino acid sequence is as shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant vector, and the recombinant vector contains the DNA sequence shown in SEQ ID NO.1.

[0010] The present invention also provides a recombinant strain, and the recombinant strain contains the above-mentioned recombinant vector.

[0011] The present invention also provides primers for amplifying the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene, and the sequences of the primer pairs used are as shown in SEQ ID NO.3-4:

[0012] Tth-1: 5’-CAGTACAGCTTGAGTGTC-3’

[0013] Tth-2: 5’-TTAAGCCCTGAACTTAACAGATCTCC-3’.

[0014] The present invention also provides the application of the salt- and ethanol-tolerant β-1,3-1,4-glucanase in the brewing industry.

[0015] The beneficial effects of the present invention are as follows: The β-1,3-1,4-glucanase expressed by the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene provided by the present invention shows the maximum activity at a pH value of 5.0 and 50 °C, and the enzyme activity of this β-1,3-1,4-glucanase remains about 94%, 72% and 86% respectively at pH 4, 5 and 6, showing significant tolerance to acidic environments; it has good stability after incubation at 20-40 °C for 1 hour, and the activity remains above 80%. At the same time, this β-1,3-1,4-glucanase is ethanol-tolerant and can still maintain >70% activity in the presence of 0-20% ethanol. The research found that 1 mM Mg 2+ , NH4 + , Li + , Co 2+ , Ba 2+, SDS and EDTA can significantly improve the enzyme activity of this β-1,3-1,4-glucanase, and the improvement rates reach 18.8%, 15.5%, 16.7%, 19.1%, 15.0%, 37.1% and 26.8% respectively. However, this β-1,3-1,4-glucanase is partially inhibited by 5 mM of these metal ions. In addition, this β-1,3-1,4-glucanase is 3+ sensitive to Al 2+ , Mn 2+ , Cu 2+ , Pb 2+ , especially Fe 3+ . The inhibition rates of its activity on β-1,3-1,4-glucanase are as high as 89% (1 mM) and 80% (5 mM) respectively. This β-1,3-1,4-glucanase has a high affinity for the substrate barley β-glucan, and its K m value and V max value are 4.61 mg / mL and 2670.9 μmol / min / mg respectively. On the other hand, adding this β-1,3-1,4-glucanase during the saccharification process can significantly reduce the viscosity by 8.69% and shorten the filtration time by 39.25%. The above characteristics make the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase of the present invention have greater advantages compared with the existing β-1,3-1,4-glucanase, and can more effectively degrade β-glucanase, having a broader application prospect in the food processing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the PCR verification diagram of the recombinant strain TaGlu16B-X33;

[0017] Figure 2 It is the schematic diagram of the enzyme activity of the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B with barley β-glucan as the substrate;

[0018] Figure 3 It is the SDS-PAGE protein electrophoresis diagram of the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0019] Figure 4 It is the SDS-PAGE protein electrophoresis diagram of the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B after deglycosylation;

[0020] Figure 5 It is the optimal pH result diagram of the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0021] Figure 6pH stability results of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0022] Figure 7 Optimum temperature results of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0023] Figure 8 Thermal stability results of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0024] Figure 9 Effect of NaCl concentration on the β-1,3-1,4-glucanase activity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0025] Figure 10 Effect of ethanol concentration on the β-1,3-1,4-glucanase activity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B;

[0026] Figure 11 Kinetic parameter graph of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B using barley β-glucan as substrate. Detailed implementation mode

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0028] The test methods without specific experimental conditions mentioned below are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are materials and reagents obtained from commercial channels.

[0029] 1. Strain: Trichoderma asperellum ND-1 (GenBank accession number: MH496612) was isolated from a soil sample collected in Chifeng (Inner Mongolia, China).

[0030] 2. Vector: The Pichia pastoris X-33 and pPICZαA vector used for protein production were purchased from Invitrogen.

[0031] 3. Matrix: Barley β-glucan, beech xylan, and oligosaccharide standards were purchased from Megazyme (Ireland). Guar gum, p-nitrophenyl-D-cellobioside (pNPC), locust bean gum, and sodium carboxymethyl cellulose were purchased from Sigma-Aldrich (USA). Silica gel 60F254 was from Merck (Germany).

[0032] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0033] Example 1: Preparation of salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B

[0034] The salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B was found in the genome of T. asperellum ND-1 (GenBank accession number: MH496612) isolated from a soil sample collected from Chifeng (Inner Mongolia Autonomous Region, China). The protein encoded by the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B was analyzed using the SignalP server (http: / / www.cbs.dtu.dk / services / signalP), and a signal peptide containing 22 amino acids (MYTSAGITFSLGALLATGAANA) was found. A primer for amplifying the β-1,3-1,4-glucanase gene (without the signal peptide) was used, and the sequences of the primer pair are shown in SEQ ID NO.3-4:

[0035] Tth-1 (SEQ ID NO.3): 5’-CAGTACAGCTTGAGTGTC-3’

[0036] Tth-2 (SEQ ID NO.4): 5’-TTAAGCCCTGAACTTAACAGATCTCC-3’.

[0037] The natural gene sequence of the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B is shown in SEQ ID NO:1, specifically:

[0038]

[0039] Construction of Recombinant Strain in Example 2

[0040] To increase the yield of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B, the natural gene sequence of the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B was codon-optimized and synthesized by Beijing Zixi International Biotechnology Research Co., Ltd., and the optimized β-1,3-1,4-glucanase gene Glu16B-opt was obtained. The similarity between the optimized gene Glu16B-opt and the natural gene was 74.7%. Subsequently, the optimized β-1,3-1,4-glucanase gene Glu16B-opt was double-digested with restriction enzymes EcoR I and Xba I and ligated to the double-digested vector pPICZαA. The obtained plasmid was named pPICZαp-Glu16B-opt as the recombinant vector and confirmed by DNA sequencing.

[0041] The salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B-opt prepared in Example 1 was heterologously expressed in Pichia pastoris X-33. The recombinant vector pPICZαp-Glu16B-opt was linearized with restriction enzyme Sac I, concentrated and then electrotransformed into Pichia pastoris X-33, and cultured and purified on a YPDS plate containing 100 μg / mL Zeocin resistance to obtain a recombinant strain, named TaGlu16B-X33. The recombinant strain TaGlu16B-X33 was verified using primers AOX-F / AOX-R and the PCR reaction system, and the verification results are as Figure 1 shown, where M represents the standard molecular weight of nucleic acids, 1 represents the negative control group (wild-type Pichia pastoris X-33), 2 represents the positive control group (recombinant vector pPICZα-Glu16B-opt), and 3 represents the PCR result using the genome of the recombinant strain TaGlu16B-X33 as the template. The appearance of two bands indicates that the recombinant strain TaGlu16B-X33 contains the salt- and ethanol-tolerant β-1,3-1,4-glucanase gene Glu16B, that is, the recombinant strain TaGlu16B-X33 was successfully constructed.

[0042] The primers AOX-F / AOX-R are shown in Table 1. The PCR reaction system is shown in Table 2.

[0043] Table 1: Primers AOX-F / AOX-R

[0044]

[0045] Table 2: PCR Reaction System

[0046]

[0047] After the PCR cycle was completed, 1% agarose gel electrophoresis was performed, and positive recombinant strains were screened according to the size of the amplification products.

[0048] Example 3: Expression of recombinant strain TaGlu16B-X33

[0049] The recombinant strain TaGlu16B-X33 constructed in Example 2 was pre-cultured in 5 mL of YPD (28 °C, 12 hours, 200 rpm), and then transferred to a complex medium containing methanol (BMMY) for induction. After 6 days, the enzyme expression was evaluated using the Bradford method and confirmed by SDS-PAGE. The amino acid sequence of the protein of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B is shown in SEQ ID NO.2, specifically:

[0050] Met Tyr Thr Ser Ala Gly Ile Thr Phe Ser Leu Gly Ala Leu Leu Ala Thr Gly Ala Ala Asn Ala Gln Tyr Ser Leu Ser Val Thr Tyr Asp Ala Ser Asn Phe Phe Ser Ser Phe Asp Phe Phe Thr Asp Ser Asp Pro Thr His Gly Phe Val Gln Tyr Val Asp Gly Asn Thr Ala Asn Thr Leu Asn Leu Thr Ser Thr Leu Thr Gly Ser Val Ile Met Gly Val Asp Ser Thr Glu Leu Asn Pro Ala Asn Gly Arg Lys Ser Val Arg Val Thr Ser Gln Gln Ser Phe Asn His Gly Leu Phe Ile Ala Asp Ile Ala His Met Pro Gly Ser Ile Cys Gly Thr Trp Pro Ala Phe Trp Met Val Gly Pro Asn Trp Pro Asn Ala Gly Glu Ile Asp Ile Ile Glu Gly Val Asn Thr Gln Thr Ser Asp Ser Ile Thr Leu His Thr Ser Ala Gly Phe Ser Val Ser Asn Asp Gly Ser Ser Ser Gly Thr Gln Leu Leu Gly Ser Asp Cys Glu Gly Asn Ser Gly Cys Ser Gln Thr Thr Ser Asp Asn Gln Asn Tyr Gly Asp Gly Phe Asn Ala Ile Asn Gly Gly Val Tyr Ala Thr Glu Trp Thr Ser Asp His Ile Ser Val Trp Phe Phe Pro Arg Thr Asn Ile Pro Ser Asp Ile Ser Gly Gly Asn Pro Asn Pro Ser Gly Trp Gly Ser Pro Leu Ala Gln Phe Asn Pro Thr Asp Gly Ser Ser Ile Asp AspHis Phe Gln Asn Asn Gln Leu Val Phe Asp ThrThr Phe Cys Gly Asp Trp Ala Gly Ser Ser Asn Val Trp Asn Ser Asn Ser Glu CysSer Ala Leu Ala Ser Thr Cys Gln Glu Tyr Val Ser Asn Asn Pro Ala Ala Phe ValAsp Ala Phe Trp Ser Val Asn Ser Ile Lys Val Tyr Asp Gln Ala Ser Asn Ala ThrVal Lys Ala Thr Gly Pro Arg Arg Ser Val Lys Phe Arg Ala

[0051] After inducing the recombinant strain TaGlu16B-X33 with 1% (v / v) methanol for 6 days, the results were as Figure 2 shown. The recombinant strain TaGlu16B-X33 obtained the highest β-1,3-1,4-glucanase activity (326.25 ± 10.29 U / mL) on the 5th day. Its enzyme activity was comparable to or higher than that of β-1,3-1,4-glucanase from Cellulomonas succinogenes (432 U / mL) and Bacillus licheniformis EGW039 (67.9 U / mL).

[0052] As Figure 3 shown, SDS-PAGE analysis was performed on the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B expressed by the recombinant strain TaGlu16B-X33. Lane M: Protein standard molecular weight, Lane 1: Fermentation supernatant of TaGlu16B-X33. The results showed that the molecular weight of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B was approximately 50 kDa, about 15 kDa higher than its theoretical value (35.5 kDa), which might be due to glycosylation modification. Therefore, PNGase F was used to explore the effect of glycosylation on the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B. A mixture of denaturing buffer (pH 4.8) and 500 μg TaGlu16B was boiled and digested with PNGase F (10 U) at 37 °C for 3 hours, and the deglycosylated salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B and the original salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B were identified by SDS-PAGE. As Figure 4As shown in the figure, where lane M: protein standard molecular weight; lane 1: TaGlu16B; lane 2: deglycosylated TaGlu16B; lane 3: deglycosylase PNGase F. The results showed that the molecular weight of deglycosylated TaGlu16B was approximately 36 kDa, which was similar to the β-1,3-1,4-glucanase of Rhizobium and Bacillus licheniformis EGW039 in the literature.

[0053] Example 4: Enzymatic property analysis of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B

[0054] The enzyme activity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B was determined by 3,5-dinitrosalicylic acid (DNS). The reaction contained 50 μL of enzyme and 150 μL of 10 mg / mL barley β-glucan dissolved in 50 mM sodium citrate buffer (pH 5.0). Then 150 μL of DNS was added, and the reducing sugar was measured at a wavelength of 540 nm after boiling at 100 °C for 5 minutes. The definition of the activity unit (U) is that the enzyme produces 1 μmol of glucose equivalent per minute.

[0055] (1) Determination of the optimal pH

[0056] Prepare 100 mL each of 3 buffers with different pH values of 50 mM. The 3 buffers are glycine-hydrochloric acid buffer (pH 2.0 - 3.0), citric acid-sodium citrate buffer (pH 4.0 - 5.0), and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0 - 8.0). Use the 3 buffers to prepare 50 μL of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B with a concentration of 0.131 μg / mL and 150 μL of 5 mg / mL barley β-glucan respectively. The reaction was incubated at 50 °C for 10 min, and the measurement was taken at 540 nm. Take the maximum enzyme activity as 100%, and measure the relative enzyme activity under other pH value conditions. Each group was designed with three replicates.

[0057] As Figure 5 shown, the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B had the highest activity at pH 5.0, and still maintained about 60% of the activity at pH 4.0, indicating that the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B was eosinophilic.

[0058] (2) pH stability

[0059] Dilute the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B with three buffers of different pH values, incubate the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B at 4 °C for 1 h, and then measure the enzyme activity after 1 h of incubation with 5 mg / mL barley β-glucan as the substrate at a temperature of 50 °C and pH 4.0. Take the enzyme activity directly measured under different pH conditions (incubation for 0 h) as 100%, and calculate the relative enzyme activity under different pH conditions. Design three replicates for each group.

[0060] As Figure 6 shown, after pretreatment at 25 °C for 1 h, the enzyme activities of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B were maintained at approximately 94%, 72%, and 86% at pH values of 4.0, 5.0, and 6.0, respectively, showing significant tolerance to acidic environments.

[0061] (3) Determination of the optimal temperature

[0062] Prepare the corresponding substrate and diluted enzyme solution with a buffer of the optimal pH 5.0, and measure the enzyme activities at 20 - 80 °C according to the method described for the optimal pH 5.0 in Example 4. Take the maximum enzyme activity as 100% and measure the relative enzyme activities at other temperature conditions. Design three replicates for each group.

[0063] As Figure 7 shown, the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B showed the highest activity at 50 °C. However, when the temperature exceeded 55 °C, the activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B decreased rapidly.

[0064] (4) Temperature stability

[0065] Incubate the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B at different temperatures (20 - 80 °C) for 1 h, and then measure the enzyme activity at a temperature of 50 °C and pH 5.0. Take the enzyme activity directly measured under different temperature conditions (incubation for 0 h) as 100%, and calculate the relative enzyme activities under different temperature conditions. Design three replicates for each group.

[0066] As Figure 8As shown, the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B has good stability after incubation at 20 - 40 °C for 1 hour, and the activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B remains above 80%. However, the activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B only retains 18% at 80 °C, indicating that the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B is not heat-resistant.

[0067] (5) Effects of metal ions and chemical reagents on the activity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B

[0068] When determining the effects of metal ions and chemical reagents on the activity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B, the detected metal ions mainly include Ba 2+ , Mn 2+ , Co 2+ , Fe 2+ , NH4 + , Al 3+ , Li + , Ni 2+ , Ca 2+ , Mg 2+ , Pb 2+ , Zn 2+ , Cu 2+ , Cd 2+ , Fe 3+ , K + , and the chemical reagents include SDS, urea, and EDTA. Prepare 1 mM and 5 mM solutions of SDS, EDTA, urea, and different metal ions with the optimal pH buffer. Mix 1 mM and 5 mM SDS, EDTA, urea, and different metal ions with the enzyme solution in a certain proportion, mix thoroughly, incubate at 4 °C for 1 h, and examine the change in enzyme activity according to the method described in Example 4 under the optimal reaction conditions of 50 °C and pH 5.0. Take the enzyme activity measured with the enzyme solution without chemical reagents and ions as 100%, and determine the relative enzyme activity under different reagent incubation conditions. In addition, the activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B was also evaluated under the conditions of 0 - 20% (v / v) NaCl and ethanol concentrations. Each group was designed with three replicates.

[0069] The effects of metal ions and chemical reagents on the activity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B are shown in Table 3. 1 mM Mg 2+ , NH4 + , Li + , Co 2+ , Ba2+ SDS and EDTA can significantly increase the enzyme activity of TaGlu16B, and the increase rates reach 18.8%, 15.5%, 16.7%, 19.1%, 15.0%, 37.1% and 26.8% respectively. However, TaGlu16B is partially inhibited by 5 mM of these metal ions. In addition, this TaGlu16B is 3+ sensitive to Al 2+ sensitive to Mn 2+ sensitive to Cu 2+ sensitive to Pb 3+ especially sensitive to Fe, and the inhibition rates of its β-1,3-1,4-glucanase activity are as high as 89% (1 mM) and 80% (5 mM) respectively.

[0070] In addition, the β-1,3-1,4-glucanase activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B was further evaluated under different concentrations of ethanol and NaCl. The results of the effect of NaCl concentration on the β-1,3-1,4-glucanase activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B are as Figure 9 shown. In the presence of 2% and 15% NaCl, the β-1,3-1,4-glucanase activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B was significantly increased to 116.9% and 113% respectively. This shows that the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B has the characteristic of salt tolerance, and this salt tolerance is comparable to that of other β-1,3-1,4-glucanases, and even exceeds their salt tolerance, including those from Paenibacillus sp. This salt tolerance makes the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B of the present invention extremely advantageous in the production of salt products. The results of the effect of ethanol concentration on the β-1,3-1,4-glucanase activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B are as Figure 10 shown. When the ethanol concentration is 0-20%, the β-1,3-1,4-glucanase activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B exceeds 70%. This shows that the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B is an ethanol-tolerant enzyme, and the ethanol-tolerant characteristic makes the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B have potential application value in the food industry (environments with high concentrations of ethanol such as alcohol clarification and feed fermentation).

[0071] Table 3: Results of the effects of metal ions and chemical reagents on the β-1,3-1,4-glucanase activity of the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B

[0072]

[0073]

[0074] (6) Substrate Specificity and Kinetic Characteristics of Salt- and Ethanol-Tolerant β-1,3-1,4-Glucanase TaGlu16B

[0075] The substrate specificity and kinetic parameters were determined using the standard DNS method. Polysaccharides (sodium carboxymethyl cellulose, laminarin, sodium alginate, guar gum, barley β-glucan, beechwood xylan, wheat arabinoxylan, and locust bean gum) and pNPC were used as substrates, and the enzyme activities of the recombinant protein against different substrates were determined using the aforementioned method. Three replicates were designed for each group. The released reducing sugars and p-nitrophenol were detected at wavelengths of 540 nm and 405 nm, respectively. The kinetic parameters of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B against 1 - 10 mg / mL barley β-glucan were determined at 50 °C and pH 5.0, and the K m and V max were analyzed using GraphPad Prism software.

[0076] The substrate specificity of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B is shown in Table 4. The results indicate that the enzyme shows a strict preference for barley β-glucan, with an enzyme activity of 326.25 ± 10.29 (U / mL). In addition, it was noted that salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B has catalytic ability towards pNPC, with an enzyme activity of 41.73 ± 3.56 (U / mL), suggesting that salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B may have the properties of an exoglucanase.

[0077] The kinetic parameters of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B were determined using barley β-glucan at 50 °C and pH 5.0. The results are as Figure 11 shown. It was determined that the K m value and V max value of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B were 4.61 mg / mL and 2670.9 μmol / min / mg, respectively.

[0078] Table 4: Substrate Specificity of Salt- and Ethanol-Tolerant β-1,3-1,4-Glucanase TaGlu16B

[0079]

[0080] Example 5: Application of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B in the brewing industry

[0081] Put 50.0 g of ground malt sample (screened with a 0.2 mm sieve) into 200 mL of water and keep it at 45 °C for 30 minutes while stirring continuously. Subsequently, heat it up to 70 °C at a rate of 1 °C / min, then add 100 mL of water, and keep the mixture at 70 °C for 1 hour. At the beginning of saccharification, different amounts of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B (0 - 100 U / g) were added, and no salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B (0 U / g) was added as a control group. Immediately cool the mash to 20 °C in an ice-water bath to terminate the reaction. By evaluating the viscosity (η) and filtration time of the wort, it was determined whether the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B could function in the brewing industry.

[0082] The results of the effects of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B on the filtration time and viscosity of wort are shown in Table 5. Under standard saccharification conditions, after adding TaGlu16B (100 U / g malt), the viscosity decreased by 8.69, and this result is higher than that of β-1,3-1,4-glucanase from Paenibacillus barengoltzii (3.40%), β-1,3-1,4-glucanase from Rhizomucor miehei (2.61%) and Fibrobactersp. UWP2 (4.50%) in the literature. In addition, the effect of TaGlu16B (100 U / g malt) on the filtration rate decreased by 39.25%. In addition, this result is also higher than that of β-1,3-1,4-glucanase PbBglu16A (14%) from Paenibacillus barengoltzii and FsGlc16A (18.89%) of Fibrobacter UWP2 reported in the literature. Therefore, the salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B has broad application prospects in the brewing industry.

[0083] Table 5: Effects of salt- and ethanol-tolerant β-1,3-1,4-glucanase TaGlu16B on filtration time and viscosity

[0084]

[0085] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase gene, characterized in that: The DNA sequences of the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase genes are shown in SEQ ID NO.

1.

2. A salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase expressed by the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase gene according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector comprises the DNA sequence shown in SEQ ID NO.1 in claim 1.

4. A recombinant strain, characterized in that: The recombinant strain comprises the recombinant vector according to claim 3.

5. A primer for amplifying the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase gene according to claim 1, characterized in that: The sequences of the primer pairs used are shown in SEQ ID NO.3-4: Tth-1:5'-CAGTACAGCTTGAGTGTC-3' Tth-2: 5'-TTAAGCCCTGAACTTAACAGATCTCC-3'.

6. Use of the salt-tolerant and ethanol-tolerant β-1,3-1,4-glucanase according to claim 2, characterized in that: Application of the salt-resistant and ethanol-resistant beta-1,3-1,4-glucanase in the brewing industry.

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